The hepatitis C virus (HCV) is classified as part of the Flaviviridae family, and contains a single, positive strand of RNA approximately 9400 nucleotides long, encoding for at least a 3000 amino acid polyprotein, depending on the source of the viral isolate. (Choo Q-L., et al., Proc. Natl. Acad. Sci. USA 88:2451-2455, 1991; Choo Q-L., et al., Science 244:359-362, 1989; Kato N., et al., Proc. Natl. Acad. Sci. USA 87:9524-9528, 1990; Takamizawa A., et al., J. Virol. 65:1105-1113, 1991) The 5′-end of the genome encodes for the structural proteins that include the nucleocapsid protein (C), and two envelope proteins (E1, and E2/NS1), whereas the 3′-end of the genome encodes for the non-structural proteins that include the NS2, NS3, NS4, and NS5 proteins. (Miller R. H. and Purcell R. H., Proc. Natl. Acad. Sci. USA 87:2057-2061, 1990; Takeuchi K., et al., J. Gen. Virol. 71:3027-3033, 1990; Choo Q-L., et al., Proc. Natl. Acad. Sci. USA 88:2451-2455, 1991; Hijikata M., et al., Proc. Natl. Acad. Sci. USA 88:5547-5551, 1991; Takamizawa A., et al., J. Virol. 65:1105-1113, 1991; Houghton M., et al., Hepatology 14:381-388, 1991) The hepatitis C virus is the main causative agent of non-A, non-B hepatitis, and plays a major role in the development of chronic liver disease, liver cirrhosis, and hepatocellular carcinoma worldwide. (Choo Q-L, et al., Proc. Natl. Acad. Sci. USA 88:2451-2455, 1991) Since there is no vaccine and no effective therapy for HCV induced disease, diagnosis and prevention of infection are issues of major public health importance.
In an effort to improve the efficiency of HCV diagnosis, many antigenic regions have been identified along the HCV genome, and used to develop three generations of enzyme immunoassays for the detection of anti-HCV activity in human sera. (Kuo et al., Science 244:362-364, 1989; Chien D. Y., et al., Proc. Natl. Acad. Sci. USA 89:10011-10015, 1992) Each successive generation represented an improvement in both the sensitivity and specificity of the enzyme immunoassay (EIA) by adding more antigenic regions to the assay. The first generation of enzyme immunoassays relied on the detection of antibodies to a region within a non-structural protein, 5-1-1. Second and third generation assays were based on the detection of antibodies against the recombinant 22 kDa core or nucleocapsid (NC) protein and several recombinant proteins derived from non-structural regions of the viral polyprotein (NS3, NS4, NS5).
Although the improvements in the specificity and sensitivity of the EIAs have resulted in a reduction in the number of new HCV infections (Alter H. J., Blood 85(7):1681-1695, 1995), many investigators have indicated that the current versions still require further development. (Tobler L. H., et al., Transfusion 34:130-134, 1994; Courouce A. M., et al., Transfusion 34:790-795, 1994; Damen M., et al., Transfusion 35:745-749, 1995; Feucht H. H., et al., J. Med Virol. 48:184-190, 1995; Bar-Shany S., et al., Inetrnl. J. Epi. 25:674-677, 1996; Dhaliwal S. K., et al., J. Med. Virol. 48:184-190, 1996; Pawlotsky J. M., et al., J. Clin. Micro. Jan:80-83, 1996) The impetus to improve tests for detection of anti-HCV is based upon studies demonstrating that currently available EIAs have relatively poor specificities, especially in low-prevalence populations. (Alter H. J., Blood 85(7):1681-1695, 1995; Feucht H. H., et al., J. Med. Virol. 48:184-190, 1995) Additionally, even after the development of supplemental tests, such as MATRIX immunoassay (Abbott Laboratories, Abbott Park, Ill.), used to confirm EIA positive sera, 10% of specimens are still classified as indeterminate (reactive to a single antigen) following supplemental testing. (Pawlotsky J. M., et al., J. Clin. Micro. Jan:80-83, 1996) These findings might be due to testing sera during the very early stage of infection before all antibodies reach detectable levels. Alternatively, reactivity to a single antigen may be due to non-specificity of the specimen.
Another important limitation to currently available assays is the use of genotype 1 recombinant proteins as immunologic targets. Recently, it was reported that there are differences in the serologic reactivity of the current EIAs to the different HCV genotypes. (Zein N. N., et al., Mayo Clinic Proc. 70(5):449-452, 1995; Dhaliwal S. K., et al., J. Med. Virol. 48:184-190, 1996) This observation suggests that the current EIAs, which are based on type I HCV, may need to be further improved by including antigenic epitopes from different genotypes. Therefore, it is clear that there remains a strong need for an enzyme immunoassay with increased specificity and sensitivity that would react with sera infected with multiple genotypes of the hepatitis C virus.
To create an enzyme immunoassay with broad reactivity to multiple genotypes, a synthetic protein must be assembled from a long DNA fragment containing multiple antigenic epitopes. The synthesis of long artificial DNA polynucleotides has been made possible by the availability of highly efficient methods to chemically synthesize relatively short oligonucleotides. To assemble a gene from oligonucleotides, several enzymatic reactions using polymerases and/or ligases may be used. Two methods described elsewhere (Khudyakov Y. E., et al., Nucleic Acid Res. 21(11):2747-2754, 1993; Khudyakov Y. E., et al., J. Virol. 68:(11)7067-7074, 1994; and U.S. Pat. No. 5,563,032), the polymerase chain reaction (PCR) and the Exchangeable Template Reaction (ETR), have been successfully applied to assemble synthetic genes from oligonucleotides. The use of PCR, however, is disadvantageous in cases where repeated sequences are designed in the gene, while ETR can not be used to conveniently express short fragments of the synthetic gene. Therefore, a new method of assembling synthetic genes with repeated sequences that would allow for the expression of shorter fragments of the gene, would greatly facilitate the creation of a synthetic protein to be used in an improved enzyme immunoassay, in particular for the detection of HCV.